Fluoromount g mounting medium

The immunofluorescence (IF) staining was performed as described previously [13 (link),36 (link)]. Briefly, sections/membranes were washed three times with PBS pH 7.4 for 5 min, followed by blocking with 4% BSA, 0.5% saponin and 10% normal goat serum in PBS pH 7.4 overnight. The primary antibodies against FcRn (Pirbright Institute, Woking, UK) and against FCGR2/CD32 (Novus Biologicals/Bio-Techne GmbH, Wiesbaden, Germany) were diluted 1:100 in PBS pH 7.4 containing 4% BSA and 0.5% saponin and incubated for 24 to 48 h at 4 °C. Afterwards, the sections/membranes were washed again three times and incubated with the corresponding secondary antibody (Table 1, 1:500 diluted in PBS pH 7.4) for 2 h. Subsequently, three additional washing steps were performed. Nuclei were stained via DAPI (20 µg/mL in PBS, Thermo Fisher, Dreieich, Germany) for 10 min and washed in PBS pH 7.4. After three additional washing steps, slides were mounted with Fluoromount G mounting medium (Sigma-Aldrich, Taufkirchen, Germany).

Pancreas tissue was harvested from 15-, 25-, and 35-week-old male BL/6 and BL/6-CD3FLAGmIR mice after intraventricular perfusion and fixed in 10% w/v formalin at 4 °C overnight. Sections were labelled for Hematoxylin and Eosin (H&E, Thermo Fisher Scientific, Pittsburg, PA, USA) after de-paraffinization and rehydration of tissue slides. After washing, sections were mounted with Fluoromount G mounting medium (Sigma, St. Louis, MO, USA) and imaged on a Cytation 5 Tissue Imager (BioRad, Hercules, CA, USA) or an Olympus VS120 slide scanner (Olympus, Center Valley, PA, USA) on brightfield setting.